Stem cells reside in adult and embryonic tissues in a broad spectrum of developmental stages and lineages, and they are thus naturally exposed to diverse microenvironments or niches that modulate their hallmark behaviors of self-renewal and differentiation into one or more mature lineages. Within each such microenvironment, stem cells sense and process multiple biochemical and biophysical cues, which can exert redundant, competing, or orthogonal influences to collectively regulate cell fate and function. The proper presentation of these myriad regulatory signals is required for tissue development and homeostasis, and their improper appearance can potentially lead to disease. Considerable advances in the field have led to the general appreciation that soluble protein cues are important regulatory signals; however, stem cells reside in a solid phase microenvironment that also presents key regulatory information from the extracellular matrix and from neighboring cells. For example, neural stem cells (NSCs) in the adult brain are surrounded by many other cell types - including other NSCs, cells in the process of differentiating, mature neurons, astrocytes, oligodendrocytes, and endothelial cells - many of which have been implicated in conveying regulatory cues to the NSCs. However, the biology of cell-cell interactions can be challenging to control and dissect using traditional cell culture systems, as it is currently diffiult to establish and maintain a precise geometrical arrangement within an ensemble of cells for sufficient times to enable rich stem cell behaviors to emerge. We propose to develop novel culture systems to pattern one or more cell types - NSCs and other constituent cells of their niche - with high spatial precision on a substrate. First, we have recently shown that a novel trap-and-corral technology composed of microwells with a PDMS mesh to contain cells within the wells for extended time periods. We will utilize this system to study the effects of homotypic, NSC-NSC interactions on cell fate decisions. Second, we will utilize a recently developed technology that uses DNA oligonucleotide base pairing to establish a cell pattern on a surface that we have found NSCs can maintain for time periods that enable analysis of cell differentiation. We will use this technology to vary the numbers and cell types that contact a NSC and thereby analyze how the number and nature of cell-cell contacts dynamically regulate NSC fate decisions. This blend of novel biosurface patterning technologies will therefore enable current and future investigations into basic mechanisms by which cell-cell contacts in the stem cell niche regulate NSC behavior, work with both basic implications for stem cell and developmental biology and biomedical applications for the development of stem cell therapies. PUBLIC HEALTH RELEVANCE: The central goal of this application is to apply microfabrication and surface patterning technology to study and begin to elucidate mechanisms by which the cellular composition and geometrical cellular organization within a niche regulates stem cell function.